The kidney protects against sepsis by producing systemic uromodulin.

Sepsis is a significant cause of mortality in hospitalized patients. Concomitant development of acute kidney injury (AKI) increases sepsis mortality through unclear mechanisms. Although electrolyte disturbances and toxic metabolite buildup during AKI could be important, it is possible that the kidney produces a protective molecule lost during sepsis with AKI. We have previously demonstrated that systemic Tamm-Horsfall protein (THP; uromodulin), a kidney-derived protein with immunomodulatory properties, falls in AKI. Using a mouse sepsis model without severe kidney injury, we showed that the kidney increases circulating THP by enhancing the basolateral release of THP from medullary thick ascending limb cells. In patients with sepsis, changes in circulating THP were positively associated with a critical illness. THP was also found de novo in injured lungs. Genetic ablation of THP in mice led to increased mortality and bacterial burden during sepsis. Consistent with the increased bacterial burden, the presence of THP in vitro and in vivo led macrophages and monocytes to upregulate a transcriptional program promoting cell migration, phagocytosis, and chemotaxis, and treatment of macrophages with purified THP increases phagocytosis. Rescue of septic THP-/- mice with exogenous systemic THP improved survival. Together, these findings suggest that through releasing THP, the kidney modulates the immune response in sepsis by enhancing mononuclear phagocyte function, and systemic THP has therapeutic potential in sepsis.NEW & NOTEWORTHY Specific therapies to improve outcomes in sepsis with kidney injury have been limited by an unclear understanding of how kidney injury increases sepsis mortality. Here, we identified Tamm-Horsfall protein, known to protect in ischemic acute kidney injury, as protective in preclinical sepsis models. Tamm-Horsfall protein also increased in clinical sepsis without severe kidney injury and concentrated in injured organs. Further study could lead to novel sepsis therapeutics.

HIV Infection Is Associated with Loss of Anti-Inflammatory Alveolar Macrophages.

HIV type 1 is associated with pulmonary dysfunction that is exacerbated by cigarette smoke. Alveolar macrophages (AM) are the most prominent immune cell in the alveolar space. These cells play an important role in clearing inhaled pathogens and regulating the inflammatory environment; however, how HIV infection impacts AM phenotype and function is not well understood, in part because of their autofluorescence and the absence of well-defined surface markers. The main aim of this study was to evaluate the impact of HIV infection on human AM and to compare the effect of smoking on their phenotype and function. Time-of-flight mass cytometry and RNA sequencing were used to characterize macrophages from human bronchoalveolar lavage of HIV-infected and -uninfected smokers and nonsmokers. We found that the frequency of CD163+ anti-inflammatory AM was decreased, whereas CD163-CCR7+ proinflammatory AM were increased in HIV infection. HIV-mediated proinflammatory polarization was associated with increased levels of inflammatory cytokines and macrophage activation. Conversely, smoking heightened the inflammatory response evident by change in the expression of CXCR4 and TLR4. Altogether, these findings suggest that HIV infection, along with cigarette smoke, favors a proinflammatory macrophage phenotype associated with enhanced expression of inflammatory molecules. Further, this study highlights time-of-flight mass cytometry as a reliable method for immunophenotyping the highly autofluorescent cells present in the bronchoalveolar lavage of cigarette smokers.

Exact variance component tests for longitudinal microbiome studies.

In metagenomic studies, testing the association between microbiome composition and clinical outcomes translates to testing the nullity of variance components. Motivated by a lung human immunodeficiency virus (HIV) microbiome project, we study longitudinal microbiome data by using variance component models with more than two variance components. Current testing strategies only apply to models with exactly two variance components and when sample sizes are large. Therefore, they are not applicable to longitudinal microbiome studies. In this paper, we propose exact tests (score test, likelihood ratio test, and restricted likelihood ratio test) to (a) test the association of the overall microbiome composition in a longitudinal design and (b) detect the association of one specific microbiome cluster while adjusting for the effects from related clusters. Our approach combines the exact tests for null hypothesis with a single variance component with a strategy of reducing multiple variance components to a single one. Simulation studies demonstrate that our method has a correct type I error rate and superior power compared to existing methods at small sample sizes and weak signals. Finally, we apply our method to a longitudinal pulmonary microbiome study of HIV-infected patients and reveal two interesting genera Prevotella and Veillonella associated with forced vital capacity. Our findings shed light on the impact of the lung microbiome on HIV complexities. The method is implemented in the open-source, high-performance computing language Julia and is freely available at https://github.com/JingZhai63/VCmicrobiome.

Biomarkers of Delirium Duration and Delirium Severity in the ICU.

OBJECTIVES: Both delirium duration and delirium severity are associated with adverse patient outcomes. Serum biomarkers associated with delirium duration and delirium severity in ICU patients have not been reliably identified. We conducted our study to identify peripheral biomarkers representing systemic inflammation, impaired neuroprotection, and astrocyte activation associated with delirium duration, delirium severity, and in-hospital mortality. DESIGN: Observational study. SETTING: Three Indianapolis hospitals. PATIENTS: Three-hundred twenty-one critically ill delirious patients. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: We analyzed the associations between biomarkers collected at delirium onset and delirium-/coma-free days assessed through Richmond Agitation-Sedation Scale/Confusion Assessment Method for the ICU, delirium severity assessed through Confusion Assessment Method for the ICU-7, and in-hospital mortality. After adjusting for age, gender, Acute Physiology and Chronic Health Evaluation II score, Charlson comorbidity score, sepsis diagnosis and study intervention group, interleukin-6, -8, and -10, tumor necrosis factor-α, C-reactive protein, and S-100ß levels in quartile 4 were negatively associated with delirium-/coma-free days by 1 week and 30 days post enrollment. Insulin-like growth factor-1 levels in quartile 4 were not associated with delirium-/coma-free days at both time points. Interleukin-6, -8, and -10, tumor necrosis factor-α, C-reactive protein, and S-100ß levels in quartile 4 were also associated with delirium severity by 1 week. At hospital discharge, interleukin-6, -8, and -10 retained the association but tumor necrosis factor-α, C-reactive protein, and S-100ß lost their associations with delirium severity. Insulin-like growth factor-1 levels in quartile 4 were not associated with delirium severity at both time points. Interleukin-8 and S-100ß levels in quartile 4 were also associated with higher in-hospital mortality. Interleukin-6 and -10, tumor necrosis factor-α, and insulin-like growth factor-1 were not found to be associated with in-hospital mortality. CONCLUSIONS: Biomarkers of systemic inflammation and those for astrocyte and glial activation were associated with longer delirium duration, higher delirium severity, and in-hospital mortality. Utility of these biomarkers early in delirium onset to identify patients at a higher risk of severe and prolonged delirium, and delirium related complications during hospitalization needs to be explored in future studies.

Contribution of the anaphylatoxin receptors, C3aR and C5aR, to the pathogenesis of pulmonary fibrosis.

Complement activation, an integral arm of innate immunity, may be the critical link to the pathogenesis of idiopathic pulmonary fibrosis (IPF). Whereas we have previously reported elevated anaphylatoxins-complement component 3a (C3a) and complement component 5a (C5a)-in IPF, which interact with TGF-ß and augment epithelial injury in vitro, their role in IPF pathogenesis remains unclear. The objective of the current study is to determine the mechanistic role of the binding of C3a/C5a to their respective receptors (C3aR and C5aR) in the progression of lung fibrosis. In normal primary human fetal lung fibroblasts, C3a and C5a induces mesenchymal activation, matrix synthesis, and the expression of their respective receptors. We investigated the role of C3aR and C5aR in lung fibrosis by using bleomycin-injured mice with fibrotic lungs, elevated local C3a and C5a, and overexpression of their receptors via pharmacologic and RNA interference interventions. Histopathologic examination revealed an arrest in disease progression and attenuated lung collagen deposition (Masson's trichrome, hydroxyproline, collagen type I α 1 chain, and collagen type I α 2 chain). Pharmacologic or RNA interference-specific interventions suppressed complement activation (C3a and C5a) and soluble terminal complement complex formation (C5b-9) locally and active TGF-ß1 systemically. C3aR/C5aR antagonists suppressed local mRNA expressions of tgfb2, tgfbr1/2, ltbp1/2, serpine1, tsp1, bmp1/4, pdgfbb, igf1, but restored the proteoglycan, dcn Clinically, compared with pathologically normal human subjects, patients with IPF presented local induction of C5aR, local and systemic induction of soluble C5b-9, and amplified expression of C3aR/C5aR in lesions. The blockade of C3aR and C5aR arrested the progression of fibrosis by attenuating local complement activation and TGF-ß/bone morphologic protein signaling as well as restoring decorin, which suggests a promising therapeutic strategy for patients with IPF.-Gu, H., Fisher, A. J., Mickler, E. A., Duerson, F., III, Cummings, O. W., Peters-Golden, M., Twigg, H. L., III, Woodruff, T. M., Wilkes, D. S., Vittal, R. Contribution of the anaphylatoxin receptors, C3aR and C5aR, to the pathogenesis of pulmonary fibrosis.

Effect of Advanced HIV Infection on the Respiratory Microbiome.

RATIONALE: Previous work found the lung microbiome in healthy subjects infected with HIV was similar to that in uninfected subjects. We hypothesized the lung microbiome from subjects infected with HIV with more advanced disease would differ from that of an uninfected control population. OBJECTIVES: To measure the lung microbiome in an HIV-infected population with advanced disease. METHODS: 16s RNA gene sequencing was performed on acellular bronchoalveolar lavage (BAL) fluid from 30 subjects infected with HIV with advanced disease (baseline mean CD4 count, 262 cells/mm(3)) before and up to 3 years after starting highly active antiretroviral therapy (HAART) and compared with 22 uninfected control subjects. MEASUREMENTS AND MAIN RESULTS: The lung microbiome in subjects infected with HIV with advanced disease demonstrated decreased alpha diversity (richness and diversity) and greater beta diversity compared with uninfected BAL. Differences improved with HAART, but still persisted up to 3 years after starting therapy. Population dispersion in the group infected with HIV was significantly greater than in the uninfected cohort and declined after treatment. There were differences in the relative abundance of some bacteria between the two groups at baseline and after 1 year of therapy. After 1 year on HAART, HIV BAL contained an increased abundance of Prevotella and Veillonella, bacteria previously associated with lung inflammation. CONCLUSIONS: The lung microbiome in subjects infected with HIV with advanced disease is altered compared with an uninfected population both in diversity and bacterial composition. Differences remain up to 3 years after starting HAART. We speculate an altered lung microbiome in HIV infection may contribute to chronic inflammation and lung complications seen in the HAART era.

Household air pollution and the lung microbiome of healthy adults in Malawi: a cross-sectional study.

BACKGROUND: Domestic combustion of biomass fuels, such as wood, charcoal, crop residue and dung causes Household Air Pollution (HAP). These inhaled particulates affect more than half of the world's population, causing respiratory problems such as infection and inflammatory lung disease. We examined whether the presence of black carbon in alveolar macrophages was associated with alterations in the lung microbiome in a Malawi population. METHODS: Bronchoalveolar lavage samples from 44 healthy adults were sequenced using 16S rDNA amplification to assess microbial diversity, richness and relative taxa abundance. Individuals were classified as high or low particulate exposure as determined by questionnaire and the percentage of black carbon within their alveolar macrophages. RESULTS: Subjects in the low and high particulate groups did not differ in terms of source of fuels used for cooking or lighting. There was no difference in alpha or beta diversity by particulate group. Neisseria and Streptococcus were significantly more abundant in samples from high particulate exposed individuals, and Tropheryma was found less abundant. Petrobacter abundance was higher in people using biomass fuel for household cooking and lighting, compared with exclusive use of electricity. CONCLUSIONS: Healthy adults in Malawi exposed to higher levels of particulates have higher abundances of potentially pathogenic bacteria (Streptococcus, Neisseria) within their lung microbiome. Domestic biomass fuel use was associated with an uncommon environmental bacterium (Petrobacter) associated with oil-rich niches.

Multicenter Comparison of Lung and Oral Microbiomes of HIV-infected and HIV-uninfected Individuals.

RATIONALE: Improved understanding of the lung microbiome in HIV-infected individuals could lead to better strategies for diagnosis, therapy, and prophylaxis of HIV-associated pneumonias. Differences in the oral and lung microbiomes in HIV-infected and HIV-uninfected individuals are not well defined. Whether highly active antiretroviral therapy influences these microbiomes is unclear. OBJECTIVES: We determined whether oral and lung microbiomes differed in clinically healthy groups of HIV-infected and HIV-uninfected subjects. METHODS: Participating sites in the Lung HIV Microbiome Project contributed bacterial 16S rRNA sequencing data from oral washes and bronchoalveolar lavages (BALs) obtained from HIV-uninfected individuals (n = 86), HIV-infected individuals who were treatment naive (n = 18), and HIV-infected individuals receiving antiretroviral therapy (n = 38). MEASUREMENTS AND MAIN RESULTS: Microbial populations differed in the oral washes among the subject groups (Streptococcus, Actinomyces, Rothia, and Atopobium), but there were no individual taxa that differed among the BALs. Comparison of oral washes and BALs demonstrated similar patterns from HIV-uninfected individuals and HIV-infected individuals receiving antiretroviral therapy, with multiple taxa differing in abundance. The pattern observed from HIV-infected individuals who were treatment naive differed from the other two groups, with differences limited to Veillonella, Rothia, and Granulicatella. CD4 cell counts did not influence the oral or BAL microbiome in these relatively healthy, HIV-infected subjects. CONCLUSIONS: The overall similarity of the microbiomes in participants with and without HIV infection was unexpected, because HIV-infected individuals with relatively preserved CD4 cell counts are at higher risk for lower respiratory tract infections, indicating impaired local immune function.

Comparison of the respiratory microbiome in healthy nonsmokers and smokers.

RATIONALE: Results from 16S rDNA-encoding gene sequence-based, culture-independent techniques have led to conflicting conclusions about the composition of the lower respiratory tract microbiome. OBJECTIVES: To compare the microbiome of the upper and lower respiratory tract in healthy HIV-uninfected nonsmokers and smokers in a multicenter cohort. METHODS: Participants were nonsmokers and smokers without significant comorbidities. Oral washes and bronchoscopic alveolar lavages were collected in a standardized manner. Sequence analysis of bacterial 16S rRNA-encoding genes was performed, and the neutral model in community ecology was used to identify bacteria that were the most plausible members of a lung microbiome. MEASUREMENTS AND MAIN RESULTS: Sixty-four participants were enrolled. Most bacteria identified in the lung were also in the mouth, but specific bacteria such as Enterobacteriaceae, Haemophilus, Methylobacterium, and Ralstonia species were disproportionally represented in the lungs compared with values predicted by the neutral model. Tropheryma was also in the lung, but not the mouth. Mouth communities differed between nonsmokers and smokers in species such as Porphyromonas, Neisseria, and Gemella, but lung bacterial populations did not. CONCLUSIONS: This study is the largest to examine composition of the lower respiratory tract microbiome in healthy individuals and the first to use the neutral model to compare the lung to the mouth. Specific bacteria appear in significantly higher abundance in the lungs than would be expected if they originated from the mouth, demonstrating that the lung microbiome does not derive entirely from the mouth. The mouth microbiome differs in nonsmokers and smokers, but lung communities were not significantly altered by smoking.

Widespread colonization of the lung by Tropheryma whipplei in HIV infection.

RATIONALE: Lung infections caused by opportunistic or virulent pathogens are a principal cause of morbidity and mortality in HIV infection. It is unknown whether HIV infection leads to changes in basal lung microflora, which may contribute to chronic pulmonary complications that increasingly are being recognized in individuals infected with HIV. OBJECTIVES: To determine whether the immunodeficiency associated with HIV infection resulted in alteration of the lung microbiota. METHODS: We used 16S ribosomal RNA targeted pyrosequencing and shotgun metagenomic sequencing to analyze bacterial gene sequences in bronchoalveolar lavage (BAL) and mouths of 82 HIV-positive and 77 HIV-negative subjects. MEASUREMENTS AND MAIN RESULTS: Sequences representing Tropheryma whipplei, the etiologic agent of Whipple's disease, were significantly more frequent in BAL of HIV-positive compared with HIV-negative individuals. T. whipplei dominated the community (>50% of sequence reads) in 11 HIV-positive subjects, but only 1 HIV-negative individual (13.4 versus 1.3%; P = 0.0018). In 30 HIV-positive individuals sampled longitudinally, antiretroviral therapy resulted in a significantly reduced relative abundance of T. whipplei in the lung. Shotgun metagenomic sequencing was performed on eight BAL samples dominated by T. whipplei 16S ribosomal RNA. Whole genome assembly of pooled reads showed that uncultured lung-derived T. whipplei had similar gene content to two isolates obtained from subjects with Whipple's disease. CONCLUSIONS: Asymptomatic subjects with HIV infection have unexpected colonization of the lung by T. whipplei, which is reduced by effective antiretroviral therapy and merits further study for a potential pathogenic role in chronic pulmonary complications of HIV infection.

Serum proteomic analysis in esophagectomy patients with postoperative delirium: A case-control study.

BACKGROUND: Postoperative delirium occurs in up to 80% of patients undergoing esophagectomy. We performed an exploratory proteomic analysis to identify protein pathways that may be associated with delirium post-esophagectomy. OBJECTIVES: Identify proteins associated with delirium and delirium severity in a younger and higher-risk surgical population. METHODS: We performed a case-control study using blood samples collected from patients enrolled in a negative, randomized, double-blind clinical trial. English speaking adults aged 18 years or older, undergoing esophagectomy, who had blood samples obtained were included. Cases were defined by a positive delirium screen after surgery while controls were patients with negative delirium assessments. Delirium was assessed using Richmond Agitation Sedation Scale and Confusion Assessment Method for the Intensive Care Unit, and delirium severity was assessed by Delirium Rating Scale-Revised-98. Blood samples were collected pre-operatively and on post-operative day 1, and discovery proteomic analysis was performed. Between-group differences in median abundance ratios were reported using Wilcoxon-Mann-Whitney Odds (WMWodds1) test. RESULTS: 52 (26 cases, 26 controls) patients were included in the study with a mean age of 64 (SD 9.6) years, 1.9% were females and 25% were African American. The median duration of delirium was 1 day (IQR: 1-2), and the median delirium/coma duration was 2.5 days (IQR: 2-4). Two proteins with greater relative abundance ratio in patients with delirium were: Coagulation factor IX (WMWodds: 1.89 95%CI: 1.0-4.2) and mannosyl-oligosaccharide 1,2-alpha-mannosidase (WMWodds: 2.4 95%CI: 1.03-9.9). Protein abundance ratios associated with mean delirium severity at postoperative day 1 were Complement C2 (Spearman rs = -0.31, 95%CI [-0.55, -0.02]) and Mannosyl-oligosaccharide 1,2-alpha-mannosidase (rs = 0.61, 95%CI = [0.29, 0.81]). CONCLUSIONS: We identified changes in proteins associated with coagulation, inflammation, and protein handling; larger, follow-up studies are needed to confirm our hypothesis-generating findings.

Anti-cytomegalovirus antibody levels stratify human immune profiles across the lifespan.

Human cytomegalovirus (hCMV) is a ubiquitous latent persistent herpesvirus infecting 60-90% of the population worldwide. hCMV carriage in immunocompetent people is asymptomatic; thus, hCMV can be considered a component of normative aging. However, hCMV powerfully modulates many features of the immune, and likely other, systems and organs. Questions remain as to how hCMV carriage affects the human host. We used anti-CMV antibody titers as a stratifying criterion to examine the impact of "intensity" of hCMV infection as a potential biomarker of aging, inflammation, and immune homeostasis in a cohort of 247 participants stratified into younger (21-40 years) and older (> 65 years of age) groups. We showed that anti-CMV antibody titers increased with age and directly correlated to increased levels of soluble tumor necrosis factor (sTNFR) I in younger but not older participants. CD8 + cell numbers were reduced in the older group due to the loss in CD8 + T naïve (Tn) cells. In CMV carriers and, in particular, in anti-CMV Ab-high participants, this loss was mitigated or reversed by an increase in the numbers of CD8 + T effector memory (Tem) and T effector memory reexpressing CD45RA (Temra) cells. Analysis of CD38, HLA-DR, and CD57 expression revealed subset (CD4 or CD8)-specific changes that correlated with anti-CMV Ab levels. In addition, anti-CMV Ab levels predicted anti-CMV CD8 T cell responsiveness to different CMV open reading frames (ORFs) selectively in older participants, which correlated to the transcriptional order of expression of specific CMV ORFs. Implications of these results for the potential predictive value of anti-CMV Ab titers during aging are discussed.

Targeted metabolomics reveals plasma biomarkers and metabolic alterations of the aging process in healthy young and older adults.

With the exponential growth in the older population in the coming years, many studies have aimed to further investigate potential biomarkers associated with the aging process and its incumbent morbidities. Age is the largest risk factor for chronic disease, likely due to younger individuals possessing more competent adaptive metabolic networks that result in overall health and homeostasis. With aging, physiological alterations occur throughout the metabolic system that contribute to functional decline. In this cross-sectional analysis, a targeted metabolomic approach was applied to investigate the plasma metabolome of young (21-40y; n = 75) and older adults (65y + ; n = 76). A corrected general linear model (GLM) was generated, with covariates of gender, BMI, and chronic condition score (CCS), to compare the metabolome of the two populations. Among the 109 targeted metabolites, those associated with impaired fatty acid metabolism in the older population were found to be most significant: palmitic acid (p < 0.001), 3-hexenedioic acid (p < 0.001), stearic acid (p = 0.005), and decanoylcarnitine (p = 0.036). Derivatives of amino acid metabolism, 1-methlyhistidine (p = 0.035) and methylhistamine (p = 0.027), were found to be increased in the younger population and several novel metabolites were identified, such as cadaverine (p = 0.034) and 4-ethylbenzoic acid (p = 0.029). Principal component analysis was conducted and highlighted a shift in the metabolome for both groups. Receiver operating characteristic analyses of partial least squares-discriminant analysis models showed the candidate markers to be more powerful indicators of age than chronic disease. Pathway and enrichment analyses uncovered several pathways and enzymes predicted to underlie the aging process, and an integrated hypothesis describing functional characteristics of the aging process was synthesized. Compared to older participants, the young group displayed greater abundance of metabolites related to lipid and nucleotide synthesis; older participants displayed decreased fatty acid oxidation and reduced tryptophan metabolism, relative to the young group. As a result, we offer a better understanding of the aging metabolome and potentially reveal new biomarkers and predicted mechanisms for future study.

SARS-CoV-2 infection of human lung epithelial cells induces TMPRSS-mediated acute fibrin deposition.

Severe COVID-associated lung injury is a major confounding factor of hospitalizations and death with no effective treatments. Here, we describe a non-classical fibrin clotting mechanism mediated by SARS-CoV-2 infected primary lung but not other susceptible epithelial cells. This infection-induced fibrin formation is observed in all variants of SARS-CoV-2 infections, and requires thrombin but is independent of tissue factor and other classical plasma coagulation factors. While prothrombin and fibrinogen levels are elevated in acute COVID BALF samples, fibrin clotting occurs only with the presence of viral infected but not uninfected lung epithelial cells. We suggest a viral-induced coagulation mechanism, in which prothrombin is activated by infection-induced transmembrane serine proteases, such as ST14 and TMPRSS11D, on NHBE cells. Our finding reveals the inefficiency of current plasma targeted anticoagulation therapy and suggests the need to develop a viral-induced ARDS animal model for treating respiratory airways with thrombin inhibitors.

T-cell cellular stress and reticulocyte signatures, but not loss of naïve T lymphocytes, characterize severe COVID-19 in older adults.

In children and younger adults up to 39 years of age, SARS-CoV-2 usually elicits mild symptoms that resemble the common cold. Disease severity increases with age starting at 30 and reaches astounding mortality rates that are ~330 fold higher in persons above 85 years of age compared to those 18-39 years old. To understand age-specific immune pathobiology of COVID-19, we have analyzed soluble mediators, cellular phenotypes, and transcriptome from over 80 COVID-19 patients of varying ages and disease severity, carefully controlling for age as a variable. We found that reticulocyte numbers and peripheral blood transcriptional signatures robustly correlated with disease severity. By contrast, decreased numbers and proportion of naïve T-cells, reported previously as a COVID-19 severity risk factor, were found to be general features of aging and not of COVID-19 severity, as they readily occurred in older participants experiencing only mild or no disease at all. Single-cell transcriptional signatures across age and severity groups showed that severe but not moderate/mild COVID-19 causes cell stress response in different T-cell populations, and some of that stress was unique to old severe participants, suggesting that in severe disease of older adults, these defenders of the organism may be disabled from performing immune protection. These findings shed new light on interactions between age and disease severity in COVID-19.

T-cell cellular stress and reticulocyte signatures, but not loss of naïve T lymphocytes, characterize severe COVID-19 in older adults.

In children and younger adults up to 39 years of age, SARS-CoV-2 usually elicits mild symptoms that resemble the common cold. Disease severity increases with age starting at 30 and reaches astounding mortality rates that are ~330 fold higher in persons above 85 years of age compared to those 18-39 years old. To understand age-specific immune pathobiology of COVID-19 we have analyzed soluble mediators, cellular phenotypes, and transcriptome from over 80 COVID-19 patients of varying ages and disease severity, carefully controlling for age as a variable. We found that reticulocyte numbers and peripheral blood transcriptional signatures robustly correlated with disease severity. By contrast, decreased numbers and proportion of naïve T-cells, reported previously as a COVID-19 severity risk factor, were found to be general features of aging and not of COVID-19 severity, as they readily occurred in older participants experiencing only mild or no disease at all. Single-cell transcriptional signatures across age and severity groups showed that severe but not moderate/mild COVID-19 causes cell stress response in different T-cell populations, and some of that stress was unique to old severe participants, suggesting that in severe disease of older adults, these defenders of the organism may be disabled from performing immune protection. These findings shed new light on interactions between age and disease severity in COVID-19.

Sphingolipid-mediated inhibition of apoptotic cell clearance by alveolar macrophages.

A decreased clearance of apoptotic cells (efferocytosis) by alveolar macrophages (AM) may contribute to inflammation in emphysema. The up-regulation of ceramides in response to cigarette smoking (CS) has been linked to AM accumulation and increased detection of apoptotic alveolar epithelial and endothelial cells in lung parenchyma. We hypothesized that ceramides inhibit the AM phagocytosis of apoptotic cells. Release of endogenous ceramides via sphingomyelinase or exogenous ceramide treatments dose-dependently impaired apoptotic Jurkat cell phagocytosis by primary rat or human AM, irrespective of the molecular species of ceramide. Similarly, in vivo augmentation of lung ceramides via intratracheal instillation in rats significantly decreased the engulfment of instilled target apoptotic thymocytes by resident AM. The mechanism of ceramide-induced efferocytosis impairment was dependent on generation of sphingosine via ceramidase. Sphingosine treatment recapitulated the effects of ceramide, dose-dependently inhibiting apoptotic cell clearance. The effect of ceramide on efferocytosis was associated with decreased membrane ruffle formation and attenuated Rac1 plasma membrane recruitment. Constitutively active Rac1 overexpression rescued AM efferocytosis against the effects of ceramide. CS exposure significantly increased AM ceramides and recapitulated the effect of ceramides on Rac1 membrane recruitment in a sphingosine-dependent manner. Importantly, CS profoundly inhibited AM efferocytosis via ceramide-dependent sphingosine production. These results suggest that excessive lung ceramides may amplify lung injury in emphysema by causing both apoptosis of structural cells and inhibition of their clearance by AM.

Reconstitution of CD4 T cells in bronchoalveolar lavage fluid after initiation of highly active antiretroviral therapy.

The massive depletion of gastrointestinal-tract CD4 T cells is a hallmark of the acute phase of HIV infection. In contrast, the depletion of the lower-respiratory-tract mucosal CD4 T cells as measured in bronchoalveolar lavage (BAL) fluid is more moderate and similar to the depletion of CD4 T cells observed in peripheral blood (PB). To understand better the dynamics of disease pathogenesis and the potential for the reconstitution of CD4 T cells in the lung and PB following the administration of effective antiretroviral therapy, we studied cell-associated viral loads, CD4 T-cell frequencies, and phenotypic and functional profiles of antigen-specific CD4 T cells from BAL fluid and blood before and after the initiation of highly active antiretroviral therapy (HAART). The major findings to emerge were the following: (i) BAL CD4 T cells are not massively depleted or preferentially infected by HIV compared to levels for PB; (ii) BAL CD4 T cells reconstitute after the initiation of HAART, and their infection frequencies decrease; (iii) BAL CD4 T-cell reconstitution appears to occur via the local proliferation of resident BAL CD4 T cells rather than redistribution; and (iv) BAL CD4 T cells are more polyfunctional than CD4 T cells in blood, and their functional profile is relatively unchanged after the initiation of HAART. Taken together, these data suggest mechanisms for mucosal CD4 T-cell depletion and interventions that might aid in the reconstitution of mucosal CD4 T cells.